Spatio-Temporal Organisation of Genome Surveillance in Live Cells

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Surveillance of the genome, which is vital for cellular function, cancer avoidance and many aspects of development, is comprised of a series of DNA repair and damage response pathways. Defects in damage surveillance result in severe genetic disorders. The mechanisms of these pathways are understood in varying degrees of detail, and the aim of this proposal is to understand the dynamics of the protein constituents within the cell nucleus before and after different DNA damaging treatments, as well as the inter-relationships between the different pathways. Normal and characterised mutant proteins tagged with GFP and its spectral variants, are either available from the participating laboratories or will be generated as part of the proposal. Motilities of the proteins are measured using variations of fluorescence recovery after photobleaching combined with whole cell or localised irradiation with either UV light or ionizing radiation. The complementarity of the partners comes from their expertise in (1) different surveillance pathways and provision of tagged proteins; (2) advanced microscopic techniques; (3) delivery of different types of localised irradiation; (4) computer simulation. Through the integration of the different expertises, unique materials and reagents, and specialized equipment from the participating groups, the proposal forms a comprehensive and multidisciplinary approach to understanding the dynamics of genome surveillance in mammalian cells.